Extended release immunomodulatory implant to facilitate bone morphogenesis

ABSTRACT

An extended release immunomodulatory implant operatively arranged to facilitate bone morphogenesis, including an inner portion including one or more interleukins, and an outer portion including an immunomodulatory stimulant such as an antigen.

FIELD

The present disclosure relates to the nascent field of osteoimmunologyand to the field of bone morphogenesis, and more particularly to animplant that activates the innate immune system in a sequential timedfashion to initiate and enhance bone healing and regeneration or fusion.

BACKGROUND

In 2002, bone morphogenic protein (BMP) or recombinant human bonemorphogenic protein-2 (rhBMP-2) was approved by the United States Foodand Drug Administration (FDA) for fusion of the lumbar spine. Therecommended application involved applying rhBMP-2 to collagen spongesand grafting them on to spinal vertebrae to facilitate spinal fusion.

While initially deemed as safe and without complication, widespread “onlabel” and “off label” use revealed numerous complications and adverseevents including heterotrophic ossification, osteolysis, bone cystformation, dysphagia, seroma, arachnoiditis, retrograde ejaculation, andincreased neurologic deficits largely secondary to the severeinflammatory reaction that ensues when pharmacologic doses (as much as1000 times over normal) are used to potentiate what is generally awell-orchestrated biologic event, that of normal bone healing.

While it is desirable to facilitate bony fusion in many instancesrequiring orthopedic surgery, the time-honored way has been the use ofautologous bone (patient's own bone) to accomplish this endeavor. Sinceautologous bone harvesting is itself not without complication, includinghemorrhage, infection, scarring, adjacent tissue damage, and persistentpain at the harvest site, alternative measures to facilitate rapid androbust bone healing have been coveted by orthopedic surgeons to treatconditions requiring bone healing or fusion. The main desire, of course,is a product which facilitates rapid and complete healing without thedeleterious effects of autologous bone harvesting and without thenumerous complications of bone graft enhancers such as BMP. Whileallograft and xenograft bone have been used, as well as speciallyprepared forms of these products, concerns over disease transmission byprions which are resistant to thermal, chemical, and radiationsterilization techniques still are of concern.

Thus, there is a long felt need in the art and science of orthopedicsurgery for a product, means, and method for a graft or graftsfacilitating bone fusion or healing, which does not require autologousharvesting of tissue, does not require potentially disease carryingallograft or xenograft products, and does not require pharmacologicdoses of expensive biologic stimulants to orchestrate what is or shouldbe a largely natural process.

SUMMARY

According to aspects illustrated herein, there is provided an extendedrelease immunomodulatory implant operatively arranged to facilitate bonemorphogenesis, comprising an inner portion including one or moreinterleukins, and an outer portion including an immunomodulatorystimulant such as an antigen.

According to aspects illustrated herein, there is provided an extendedrelease immunomodulatory implant operatively arranged to facilitate bonemorphogenesis, comprising an implant matrix including a first material,the implant matrix including an inner portion including at least one ofinterleukin 4, interleukin 10, and interleukin 13, and an outer portionincluding at least one of lipopolysaccharide and lipoteichoic acid.

According to aspects illustrated herein, there is provided a method ofmanufacturing an extended release immunomodulatory implant operativelyarranged to facilitate bone morphogenesis, the method comprising formingan inner portion of the implant of a first material, applying at leastone of interleukin 4, interleukin 10, and interleukin 13 to the innerportion, forming an outer portion of the implant of a second material,the outer portion enclosing the inner portion, and applying at least oneof lipopolysaccharide and lipoteichoic acid to the outer portion.

According to aspects illustrated herein, there is provided an extendedrelease immunomodulatory implant operatively arranged to facilitate bonemorphogenesis, comprising an inner portion comprising an implant matrixincluding interleukin 10 and 13 at 10 ng/ml, an inner layer arrangedaround the inner portion, the inner layer comprising an implant matrixincluding interleukin 4 at 10 ng/ml, and an outer layer arranged aroundthe inner layer, the outer layer comprising an implant matrix includingan antigen or antigen mixture (for example lipopolysaccharide,lipoteichoic acid, and/or interferon gamma) at 40 ng/ml.

According to aspects illustrated herein, there is provided an extendedrelease immunomodulatory implant operatively arranged to facilitate bonemorphogenesis, comprising a core (or inner portion) comprisinghydroxyapatite, a first layer arranged around the core, the first layercomprising beta-tricalcium phosphate including one or more interleukins(for example interleukin 4, 10, and/or 13), and a second layer arrangedaround the first layer, the second layer comprising beta-tricalciumphosphate including an antigen or antigen mixture (for examplelipopolysaccharide, lipoteichoic acid, and/or interferon gamma). In someembodiments, the core, the first layer, and the second layer are 3Dprinted and include one or more predetermined porosities. For example,the core comprises a first porosity, the first layer comprises a secondporosity, and the second layer comprises a third porosity.

According to aspects illustrated herein, there is provided an extendedrelease immunomodulatory implant operatively arranged to facilitate bonemorphogenesis, comprising a first component including at least one ofinert beta-tricalcium phosphate, calcium carbonate, silicon,polylactic-co-glycolic acid, allograft bone, autograft bone, titanium,polyether ether ketone, and a mixture applied to the first component,the mixture including at least one of lipopolysaccharide andlipoteichoic acid.

It is an object of the present disclosure to provide a graft implantwhich facilitates a rapid and robust restoration, healing, or osseousunion of selected bony elements requiring fusion or bony repair torestore integrity of a bony element, repair a large bony defect, oraffix bony elements together for therapeutic purposes (i.e., bonefusion).

It is also an object of the present disclosure to not require theharvest of autologous products, such as bone cortex, bone marrow, blood,stem cells, platelets, or the like.

Additionally, it is an object of the present disclosure that noallograft, xenograft, coral, donor stem cells, or any similar productsbe used to eliminate graft to host disease transmission.

It is also an object of the present disclosure to avoid pharmacologicdoses of cellular signaling proteins or molecules, thereby reducing theprospects of deleterious side effects already known in the art andscience of bone healing.

It is also an object of the present disclosure that the graft is costeffective such that the graft implant could be utilized even indeveloping countries challenged by the ever-rising costs of modernmedicine.

It is also an object of the present disclosure to provide a patternedsequence of growth and differentiation factors such as cytokines,enzymes, immunomodulatory molecules, or similar complexes arranged in anarray to sequentially stimulate in a biomimetic fashion the normalcascade of reparative bone morphogenesis thereby facilitating, fosteringor formulating accelerated bone growth, bone formation, bone healing andbone fusion for therapeutic purposes.

To achieve these objects, an implant or graft made of porous orfibrillary hydroxyapatite or beta tricalcium phosphate with 1% siliconby weight is chosen. This implant approximates the chemical makeup ofnormal bone but is devoid of any living elements. The size or volume ofthe implant will approximate the bone void to be filled or region to begrafted to facilitate the bony fusion.

The implant or graft comprises an outer layer or layers includinglipopolysaccharide (LPS), lipoteichoic acid (LTA), interferon gamma,and/or other antigens or stimulants, which activate M0 and M1macrophages. These substances can also chemotactically attractcirculating and tissue monocytes, which in turn can be converted to M0and M1 macrophages. In some embodiments, the outer layer is affixed withLPS and/or LTA. In some embodiments, LPS and/or LTA is adsorbed on theouter layer. In some embodiments, the outer layer is saturated orimpregnated with LPS and/or LTA. In some embodiments, the LPA is derivedfrom Escherichia coli (E. coli), such as, for example, E. coli StrainO55:B5. In some embodiments, the LTA is derived from Staphylococcusaureus (S. aureus).

The implant or graft further comprises an inner layer or recessesincluding interleukin 4, 10, or 13, either individually or incombination. In some embodiments, the inner layer is affixed withinterleukin 4, 10, and/or 13. In some embodiments, interleukin 4, 10,and/or 13 is adsorbed on the inner layer. In some embodiments, the innerlayer is saturated or impregnated with interleukin 4, 10, and/or 13. Byarranging the layers such that the activating immunomodulatorysubstance(s) is outside (i.e., on the outer layer) and the cell signallymolecules are arranged inside (i.e., on the inner layer), animmunomodulatory or “smart” implant is created, and one which morenaturally replicates or coordinates normal bone healing, albeit byconvincing the body's innate immune system that a contaminated compoundfracture exists and is in need of biologic debridement and repair. Theouter layer containing immunomodulatory molecules serves to initiate theactivity of the innate immune system via pattern recognition of the LPSand/or LTA by receptors on monocytes and macrophages which bind thesemolecules. In some embodiments, alternatively, or in conjunction,interferon gamma could be used.

In some embodiments, the implant comprises a mixture of LPS and LTAranging from 1 ng/ml to 1,000 ng/ml, with the preferred embodimentincluding 40 ng/ml. In some embodiments, the implant comprisesinterferon gamma ranging from 1 ng/ml to 1,000 ng/ml, with the preferredembodiment including 200 ng/ml. In some embodiments, the implantcomprises interleukin 4 ranging from 0.1 ng/ml to 100 ng/ml with thepreferred embodiment including 10 ng/ml. In some embodiments, theimplant comprises interleukin 10 ranging from 0.1 ng/ml to 100 ng/mlwith the preferred embodiment including 10 ng/ml. In some embodiments,the implant comprises interleukin 13 ranging from 0.1 ng/ml to 100 ng/mlwith the preferred embodiment including 10 ng/ml.

Once monocytes or M0 macrophages bind to the immunomodulatory molecule,they release a cytokine(s) which attracts more monocytes and M0macrophages, which in turn are drawn toward the implant via achemotactic gradient. The immunomodulators (i.e., LPS and/or LTA) alsofacilitate the conversion of monocytes and M0 macrophages to M1macrophages often referred to as classically activated macrophages,which in turn initiate the healing process. M1 macrophages aggressivelyphagocytose immune modulatory molecules and any tissue debris they arebound to until all of the immunomodulatory molecules are gone. In thisprocess, the thin outer layer of the implant is removed by the M1macrophages exposing the deeper layer of denser beta-tricalciumphosphate and/or hydroxyapatite containing interleukins. Interleukins(particularly interleukin 4) are a potent stimulant that converts the M1(inflammatory or classically activated) macrophage into an M2(reparative or alternately activated) macrophage, which begins thereparative process. The M1 to M2 conversion is further enhanced by thelack of LPS and/or LTA and the presence of calcium ions in directing theneed for bone repair.

By layering the activating components in this fashion, the implantserves multiple purposes and rises above the level of serving as asimple tissue scaffold. Indeed, the implant attracts, activates, andultimately converts circulating or resident monocytes and macrophagesinto participating in what is essentially an artificial bone repair.

LPS, LTA, and other immunomodulators are potent stimulants of the innateimmune system. Their presence suggests an invasion by foreign organismsand they are actively phagocytized by M1 macrophages, which in turnchemotactically attract other monocytes and macrophages to the area aspart of the initial scene of fracture repair. M0 macrophages can beconverted to M1 macrophages by LPS and/or LTA; this is known as classicactivation and is routinely done to study these cells.

M1 macrophages serve to kill bacteria, remove debris, and recruitcirculating monocytes and resident tissue macrophages to do the same.They remain M1 or classically activated macrophages until the antigen(LPS and/or LTA) is gone. This stage of M1 activation is analogous tothe inflammatory stage of fracture repair. These M1 macrophages areoften known as inflammatory macrophages for this reason.

Once the antigenic threat no longer exists (i.e., the LPS and/or LTA isgone), M1 or classically activated macrophages metamorphose into M2 oralternatively activated macrophages, which are anti-inflammatory orreparative in nature. This transformation is facilitated by the presenceof interleukin 4, 10, and 13, which is released by M1 macrophages onceall antigen is disposed of and full restoration of bone and hencefracture repair is initiated, a process which is enhanced by calciumions and silicon ions present in bone.

By having an implant constructed in such a layered fashion, the maximumcapacity of the immunomodulatory aspect of bone healing can be harnessedand modulated to facilitate or orchestrate a rapid and robust bonehealing process along its natural path.

Immunomodulatory molecules (LPS and/or LTA) suggest injury to the hostinvolving invasion by pathogens. Surface receptors of monocytes and M0macrophages encounter the immunomodulatory molecules and bind them totheir surfaces. Monocytes and M0 macrophages release cytokines toattract more cells (i.e., monocytes and M0 macrophages) to the area toconfront the pathogens and transform into M1 macrophages, whichaggressively phagocytose the immunomodulatory molecules and bonefragments to which they are attached until the immunomodulatorymolecules are gone. The lack of stimulation by LPS and/or LTA results inthe M1 macrophages producing interleukins, which facilitates thetransformation of M1 (inflammatory) macrophages into M2 (reparative)macrophages. In the presence of calcium ions, M1 macrophages alsorelease Oncostatin M (OSM), prostaglandin E2 (PGE2), and BPMs, which arenecessary to convert mesenchymal stem cells into osteoprogenitor cells,which invade the inner portion of the implant and initiate andcoordinate bone morphogenesis.

For example, the implant is laid on the surface of two bony elementsthat have been decorticated in preparation for fusion. Wound blood andserum elute LPS from the surface or outer layer of the implant signalingthe monocytes in blood to produce cytokines that attract additionalmonocytes and macrophages (M0 macrophages) to the area. Theseclassically activated (M0) macrophages begin the process of neutralizingthe bacterial invaders (antigen) by converting to M1 macrophages, aswill be discussed in greater detail below, and remain activated untilall antigen is removed from the outer layer of the implant.

Once the antigen load is negligible, classically activated M1macrophages metamorphose into alternatively activated M2 macrophages,which are key in tissue repair. The activity of M2 macrophages isenhanced by interleukins 4, 10, and 13, which are encountered in thedeeper layers or inner layer of the implant and a full repair process isinstigated. M2 macrophages, in turn, activate adjacent tissue dendriticcells, mesenchymal stem cells, and osteoprogenitor cells, which becomemature osteoblasts so that bony repair or fusion can rapidly progress.

Since avirulent activation of the immunomodulatory pathway was used, thedeleterious effects of a true infection are avoided, much like avaccination, with the killed or attenuated viruses having activated theadaptive immune system without having to suffer the disease. Once theinnate immune system is activated and the cells come in contact with thecalcium in the implant or graft, a normal healing process occurs withall of the necessary natural cell signals and the correct amounts neededto effect rapid bone morphogenesis and bone healing or fusion. Naturalbone resorption, reconstitution, and osseointegration of the graftimplant thereby occurs.

According to aspects illustrated herein, there is provided a syntheticimplant of porous hydroxyapatite and/or beta-tricalcium phosphatecontaining immunomodulators which activate the innate immune system in asequential timed fashion thereby initiating and enhancing bone healingor fusion, for example, in humans and animals.

According to aspects illustrated herein, there is provided an implantcomprising a solid, particulate, and/or fibrillary hydroxyapatite and/orbeta-tricalcium phosphate. The implant comprising an outer portionincluding absorbed or adsorbed antigen(s) of LPS and/or LTA, whichactivates the innate immune system and particularly M0 or M1macrophages, which can instigate bone morphogenesis and repair. Itshould be appreciated that other antigens suitable for activating theinnate immune system may be used, and that this disclosure should not belimited to only the use of LPS and LTA. Furthermore, a concentration ofantigen suitable to classically activate macrophages could be used. Insome embodiments, interferon gamma may be used to activate the innateimmune system.

Once the implant activates the innate immune system, all of the naturalproducts necessary to affect bone morphogenesis or bone healing areactivated and brought to the surgical site through natural cellsignaling. Upon removal of the antigen load from the surface or outerportion of the implant, classically activated M1 macrophages transformto reparative or M2 macrophages. This conversion is more rapidlyfacilitated by interleukin 4, 10, and/or 13 encountered in deeper layersor an inner portion of the implant. In some embodiments, the innerlayers or portion of the implant are less porous (and more dense) thanthe outer layers or portion.

Once M2 or alternatively activated macrophage conversion occurs, fullbone morphogenesis and healing or fusion progresses until the implant istransformed into normal bone and fusion of bony elements is complete.Macrophages (M1 and M2) furthermore release cytokines (OSM, PGE2, andBMPs), which attract mesenchymal stem cells and osteoprogenitor cellsinto the graft. These cells inhabit the inner layers or portions of theimplant or graft and transform into mature osteoblasts, which facilitatebone morphogenesis. In some embodiments, the implant is 3D printed andcomprises variations in porosity of the layers, with the outer layer orportion being the most porous and the inner layer or portion being mostdense, such that structural integrity of the implant can be maintainedwhen the outer layer or portion is removed by M1 macrophages andultimately remodeled by osteoclast and osteoblast activity. In someembodiments, the implant comprises electrospun beta-tricalcium phosphatefibers, the porosity of which can be varied by their compactness.

The implant of the present invention adds another dimension to what isalready known. Since the LPS and/or LTA elutes from the surface of theimplant, and since the elutant has the capacity to attract monocytes andM0 macrophages chemotactically, it becomes “osteopreparatory” by virtueof direct chemotaxis by the elutant and by virtue of the fact that anymonocytes, monocyte derived cells, or neutrophils that come in contactwith the antigen will further release cytokines to attract moremonocytes and M0 macrophages and enhance their conversion ortransformation to M1 macrophages as part of the innate immune responseto bodily injury and invasion by pathogens (e.g., a classic compoundfracture). By having the implant made of calcium based material, anotherrequirement is fulfilled. Calcium ions are an important signal to cellsthat bone is involved in the trauma and that bone repair will berequired. This is not critical at the macrophage level, but when themacrophages have done their work, the calcium component is important insignaling mesenchymal stem cells and osteoprogenitor cells to lay downbone.

Ultimately, it is the M2 macrophage that signals the stem and progenitorcells to begin, but calcium is needed for them to carry out normal boneformation. The M1 to M2 conversion is facilitated by the interleukins inthe inner layer (e.g., interleukins 4, 10, and/or 13) and the M2 cellsset the stage for and facilitate the performance of the mesenchymal stemcells and osteoprogenitor cells to direct morphogenesis toward matureosteoblasts, which form mature bone.

In essence, therefore, the present disclosure provide for an implantthat is unique in regenerative bone biology. The implant isosteopreparatory, osteoinductive, and osteoconductive. At present, inthe art, most bone grafts are only osteoconductive with a few, includingautograft bone, being both osteoconductive and osteoinductive.Additionally, the implant orchestrates bone morphogenesis in acontrolled, sequential, and logical fashion whereby the cellularparticipants are exploited to their maximal value to achieve rapid bonerepair and healing. No outside cells are required since the organismsown innate healing powers are directed and enhanced by employing theinnate immune system to facilitate the process. Since the innate immunesystem evolved hundreds of million years ago, this implant and processhas implications for all animals, not just humans. Any animal with abony skeleton could benefit from this healing method. Furthermore, theimplant is designed to dissolve completely, leaving no trace of itselfor its constituent components. Only normal regenerative bone is leftbehind.

Consider, therefore, the implant is placed in a surgical site having anexcoriated or prepared surface (e.g., decorticated bone). The outerlayer of the implant elutes LPS/LTA, which chemotactically attractsmonocytes, neutrophils, and M0 macrophages. The monocytes andmacrophages imbibe the LPS/LTA and destroy them with lysosomes. Thisaction releases cytokines, which further attract more monocytes and M0macrophages to the site in every increasing numbers. LPS/LTA furthertransforms monocytes and M0 macrophages into M1 or inflammatorymacrophages, also known as classically activated macrophages, whichdissolve the outer layer of the implant to which the antigen is absorbedor adsorbed. This “clean up” process continues until all of the outerlayer and the LPS/LTA associated with it is completely gone. The M1macrophages then encounter the inner layer to which is absorbed oraffixed, interleukin 4. Interleukin 4 signals the M1 macrophage orinflammatory cell to become a M2 macrophage or anti-inflammatory cell,also known as a reparative cell, or alternatively activated macrophage.The absence of LPS/LTA also favors this transformation. M2 macrophagesstart the tissue reparative process using the hydroxyapatite or betatricalcium phosphate of the inner layer as the scaffold on which therepair process using the hydroxyapatite or beta tricalcium phosphate ofthe inner layer as the scaffold on which the repair process isperformed. M2 macrophages in turn, chemotactically attract mesenchymalstem cells, dendritic cells, and osteoprogenitor cells into thescaffolding of the implant to facilitate further bone morphogenesis.Thus, the implant of the present disclosure is osteopreparatory,osteoinductive, osteoconductive, and naturally dissolves leaving purenormal regenerative bone behind.

These and other objects, features, and advantages of the presentdisclosure will become readily apparent upon a review of the followingdetailed description of the disclosure, in view of the drawings andappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments are disclosed, by way of example only, withreference to the accompanying schematic drawings in which correspondingreference symbols indicate corresponding parts, in which:

FIG. 1 is a perspective view of an implant;

FIG. 2 is a cross-sectional view of the implant taken generally alongline 2-2 in FIG. 1;

FIG. 3 is a perspective view of an implant; and,

FIG. 4 is a cross-sectional view of the implant taken generally alongline 4-4 in FIG. 3.

DETAILED DESCRIPTION

At the outset, it should be appreciated that like drawing numbers ondifferent drawing views identify identical, or functionally similar,structural elements. It is to be understood that the claims are notlimited to the disclosed aspects.

Furthermore, it is understood that this disclosure is not limited to theparticular methodology, materials and modifications described and assuch may, of course, vary. It is also understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to limit the scope of the claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure pertains. It should be understood thatany methods, devices or materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the exampleembodiments.

It should be appreciated that the term “substantially” is synonymouswith terms such as “nearly,” “very nearly,” “about,” “approximately,”“around,” “bordering on,” “close to,” “essentially,” “in theneighborhood of,” “in the vicinity of,” etc., and such terms may be usedinterchangeably as appearing in the specification and claims. It shouldbe appreciated that the term “proximate” is synonymous with terms suchas “nearby,” “close,” “adjacent,” “neighboring,” “immediate,”“adjoining,” etc., and such terms may be used interchangeably asappearing in the specification and claims. The term “approximately” isintended to mean values within ten percent of the specified value.

Referring now to the figures, FIG. 1 is a perspective view of implant10. FIG. 2 is a cross-sectional view of implant 10 taken generally alongline 2-2 in FIG. 1. Implant 10 generally comprises implant matrix 12which includes outer portion(s) or layer(s) 20 and inner portion(s) 30.Implant matrix 12 comprises inert beta-tricalcium phosphate, calciumcarbonate (CaCO3), silicon, polylactic-co-glycolic acid (PLGA), and/orhydroxyapatite. In some embodiments, implant matrix 12 consists of inertbeta-tricalcium phosphate, CaCO3, silicon, and PLGA. In someembodiments, implant matrix 12 consists of inert beta-tricalciumphosphate, CaCO3, silicon, and PLGA with silicon at 1% by weight. Insome embodiments, implant matrix 12 comprises allograft bone, autograftbone, xenograft bone, a titanium implant, a polyether ether ketone(PEEK) implant, and/or synthetic bone void filler coated and/orimpregnated with the antigen or antigen mixture (LPS, LTA, and/or othersuitable antigen) and/or interleukins 4, 10, and/or 13. By impregnated,it is meant that the implant (e.g., the inner or outer portions) isfilled, imbued, permeated, or saturated with the antigen or antigenmixture and/or interleukins. It should be appreciated that implant 10,including inner portion 30 and outer layer 20, may comprise any suitablegeometry, for example, cuboid, cube, sphere, cylinder, cone,tetrahedron, triangular prism, etc., and that the present disclosureshould not be limited to the geometric shape(s) shown in the figures.The following description should be read in view of FIGS. 1-2.

Outer portion or layer 20 comprises LPS, LTA, and/or other suitableantigen, and as discussed above, is operatively arranged to attractmonocytes and macrophages to the site via chemotaxis and initiate the M1phase of macrophages. Specifically, outer portion 20 attracts monocytesand macrophages to the site, and once there, converts M0 macrophagesinto M1 macrophages, which phagocytose outer portion 20. As such, outerportion 20 is phagocytosable, or capable of being phagocytosed. Putanother way, outer portion 20 is operatively arranged to be completelyremovable from inner portion 30 via phagocytosis. In some embodiments,outer portion 20 is operatively arranged to be partially removable frominner portion 30. In such embodiments, M1 macrophages phagocytize theantigen on or in implant matrix 12 and access inner portion(s) 30through holes and/or porosities in outer portion 20, thus leaving behindthe porous implant matrix 12 (e.g., inert beta-tricalcium phosphate,CaCO3, silicon, PLGA, hydroxyapatite, allograft bone, autograft bone,xenograft bone, a titanium implant, a PEEK implant, and/or syntheticbone void filler). In some embodiments, outer portion 20 consists of amixture of LPS and LTA. In some embodiments, outer portion 20 consistsof a mixture of 50% by weight LPS and 50% by weight LTA. In someembodiments, outer portion 20 consists of a mixture of 50% by weight LPSand 50% by weight LTA, mixed together at a concentration of 5 microgramsper liter each. In some embodiments, outer portion 20 consists of amixture of 50% by weight LPS and 50% by weight LTA, mixed together at aconcentration of 5 micrograms per liter each, wherein the LPS is derivedfrom E. coli (e.g., E. coli Strain O55:B5) and the LTA is derived fromS. aureus. In some embodiments, outer portion 20 comprises an antigen orantigen mixture of 40 ng/ml. It is also recognized that 100% LPS or 100%LTA or similar antigen can be used. Indeed, any combination ofstimulants known to specifically activate the innate immune system canbe employed.

Outer portion 20 may comprise an antigen or antigen mixture (LPS, LTA,and/or other suitable antigen) completely, or inert beta-tricalciumphosphate, CaCO3, silicon, PLGA, and/or hydroxyapatite with the antigenor antigen mixture applied thereon or embedded or injected therein. Insome embodiments, the antigen or antigen mixture (LPS, LTA, and/or othersuitable antigen) is added to implant 10 to create outer portion 20 bydipping implant 10 therein and air drying. In some embodiments, theantigen or antigen mixture (LPS, LTA, and/or other suitable antigen) isadded to implant 10 to create outer portion 20 by placing implant 10(e.g., in the wound or incision) and squirting the antigen or antigenmixture thereon.

Inner portion 30 may comprise an infusion of interleukins, and asdiscussed above, is operatively arranged to initiate the M2 phase ofmacrophages. In some embodiments, inner portion 30 comprises an infusionof capsaicin and/or interleukins. Once the M1 macrophages completelyand/or partially phagocytoses outer portion 20 (i.e., all of the LPS,LTA, and/or other suitable antigen is eaten away), the M1 macrophagesencounter inner portion 30, which comprises interleukins 4, 10, and/or13, and are converted to M2 macrophages. The M2 macrophages begin thereparative process and hence bony repair or fusion. M1 macrophages areconverted to M2 macrophages through contact with inner portion 30 (i.e.,the interleukins). Additionally, once the M1 macrophages completelyand/or partially phagocytize outer portion 20 (i.e., the antigen orantigen mixture), interleukins are released by the M1 macrophages toinitiate the M2 phase of macrophages, based in part on the dwindlingamounts of antigen left to phagocytose. Thus, implant 10 can convert M1macrophages to M2 macrophages in two ways: 1) after M1 macrophagescompletely phagocytize outer portion 20; and, 2) when M1 macrophagescome into contact with inner portion 30. In some embodiments, innerportion 30 comprises interleukin 4, 10, and/or 13.

In some embodiments, implant matrix 12 comprises beta-tricalciumphosphate, for example, 3D printed such that inner portion 30 comprisessmall pore sizes and outer portion 20 comprises larger pore sizes. Thelarger pores and reduced density of outer portion 20 allows it to beinfused with the antigen or antigen mixtures (LPS, LTA, and/or othersuitable antigen) to chemotactically attract M0 and M1 macrophages andmonocytes. The porosity and reduced density of outer portion 20 alsospeeds the process of phagocytosis, wherein the M1 macrophagesphagocytose outer portion 20 until it is gone and only inner portion 30remains. In some embodiments, and as previously discussed, outer portion20 is operatively arranged to be partially removable/dissolvable,wherein the M1 macrophages phagocytose the antigen on or in implantmatrix 12 and subsequently access inner portion 30 through holes orpores in outer portion 20. The smaller pores and increased density ofinner portion 30, which contains the interleukins, slows its dissolutionso as to remain until suitable bone growth or fusion has occurred or isoccurring. In some embodiments, inner portion(s) 30 comprises a porosityhaving an average pore size of 50-200 microns, and the outer portion(s)20 comprises a porosity having an average pore size of 200-500 microns.In some embodiments, the pores of inner portion(s) 30 and outerportion(s) 20 are interconnected by channels throughout implant 10.

Implant 10 generally acts as a dissolving implant or a time release bonefusion capsule or implant. The M0 macrophages are attracted to implant10 and converted to M1 macrophages upon arrival. The M1 macrophages“dissolve” or phagocytize outer portion 20 comprising the antigen orantigen mixture (LPS, LTA, and/or other suitable antigen) and, once thisis done, the M1 macrophages encounter the denser inner portion 30containing interleukins. Inner portion 30 containing interleukinsmodulates the transition of M1 or inflammatory macrophages to M2 oranti-inflammatory macrophages thereby facilitating the reparativeprocess (i.e., bone growth or fusion). Implant 10 is eventuallyincorporated and transformed into normal regenerative bone by activelyorchestrating the key cellular actors involved in bone healing.

FIG. 3 is a perspective view of implant 110. FIG. 4 is a cross-sectionalview of implant 110 taken generally along line 4-4 in FIG. 3. Implant110 generally comprises implant matrix 112 which includes outer layer(s)120, inner layer(s) 122, and inner portion(s) 130. Implant matrix 112comprises inert beta-tricalcium phosphate, CaCO3, silicon, PLGA, and/orhydroxyapatite. In some embodiments, implant matrix 112 consists ofinert beta-tricalcium phosphate, CaCO3, silicon, and PLGA. In someembodiments, implant matrix 112 consists of inert beta-tricalciumphosphate, CaCO3, silicon, and PLGA with silicon at 1% by weight. Insome embodiments, implant matrix 112 comprises inert beta-tricalciumphosphate or hydroxyapatite alone. In some embodiments, implant matrix112 comprises allograft bone, autograft bone, xenograft bone, a titaniumimplant, a polyether ether ketone (PEEK) implant, and/or synthetic bonevoid filler coated and/or impregnated with the antigen or antigenmixture (LPS, LTA, and/or other suitable antigen) and/or interleukins 4,10, and/or 13. By impregnated, it is meant that the implant (e.g., theinner or outer portions) is filled, imbued, permeated, or saturated withthe antigen or antigen mixture and/or interleukins. It should beappreciated that implant 110, including inner portion 130 and layers 120and 122, may comprise any suitable geometry, for example, cuboid, cube,sphere, cylinder, cone, tetrahedron, triangular prism, etc., and thatthe present disclosure should not be limited to the geometric shape(s)shown in the figures. The following description should be read in viewof FIGS. 3-4.

Outer layer 120 comprises LPS, LTA, and/or other suitable antigen, andas discussed above, is operatively arranged to attract monocytes andmacrophages to the site via chemotaxis and initiate the M1 phase ofmacrophages. Specifically, outer layer 120 attracts monocytes andmacrophages to the site, and once there, converts M0 macrophages into M1macrophages, which phagocytose outer layer 120. As such, outer layer 120is phagocytosable, or capable of being phagocytosed. Put another way,outer layer 120 is operatively arranged to be completely removable frominner layer 122 and inner portion 130 via phagocytosis. In someembodiments, outer layer 120 and/or inner layer 122 are operativelyarranged to be partially removable from inner portion 130. In suchembodiments, M1 macrophages phagocytize the antigen on or in bone matrix112 and access inner portion 130 through holes and/or porosity in outerlayer 120 and/or inner layer 122, thus leaving behind the porous bonematrix 112 (e.g., inert beta-tricalcium phosphate, CaCO3, silicon, PLGA,hydroxyapatite, allograft bone, autograft bone, xenograft bone, atitanium implant, a PEEK implant, and/or synthetic bone void filler). Insome embodiments, outer layer 120 consists of a mixture of LPS and LTA.In some embodiments, outer layer 120 consists of a mixture of 50% byweight LPS and 50% by weight LTA. In some embodiments, outer layer 120consists of a mixture of 50% by weight LPS and 50% by weight LTA, mixedtogether at a concentration of 5 micrograms per liter each. In someembodiments, outer layer 120 consists of a mixture of 50% by weight LPSand 50% by weight LTA, mixed together at a concentration of 5 microgramsper liter each, wherein the LPS is derived from E. coli (e.g., E. coliStrain O55:B5) and the LTA is derived from S. aureus. In someembodiments, outer layer 120 comprises an antigen or antigen mixture of40 ng/ml. It is recognized that 100% LPS or 100% LTA or 100% of anyantigen or any combination thereof can be chosen to optimally activatethe innate immune system.

Outer layer 120 may comprise an antigen or antigen mixture (LPS, LTA,and/or other suitable antigen) completely, or inert beta-tricalciumphosphate, CaCO3, silicon, PLGA, and/or hydroxyapatite with the antigenor antigen mixture applied thereon or embedded or injected therein. Insome embodiments, the antigen or antigen mixture (LPS, LTA, and/or othersuitable antigen) is added to implant 110 to create outer layer 120 bydipping implant 110 therein and air drying. In some embodiments, theantigen or antigen mixture (LPS, LTA, and/or other suitable antigen) isadded to implant 110 to create outer layer 120 by placing implant 110(e.g., in the wound or incision) and squirting the antigen or antigenmixture thereon. The antigen or antigen mixture could also be added atany point as part of the 3D printing process in the manufacturing of theimplant.

Inner layer 122 comprises LPS, LTA, and/or other suitable antigen, andas discussed above, is operatively arranged to attract monocytes andmacrophages to the site via chemotaxis and initiate the M1 phase ofmacrophages. Specifically, inner layer 122 attracts monocytes andmacrophages to the site, and once there, converts M0 macrophages into M1macrophages, which phagocytose outer layer 120 and subsequently innerlayer 122. As such, outer layer 120 and inner layer 122 arephagocytosable, or capable of being phagocytosed. Put another way, outerlayer 120 and inner layer 122 are operatively arranged to be completelyremovable from inner portion 130 via phagocytosis. In some embodiments,outer layer 120 and/or inner layer 122 are operatively arranged to bepartially removable from inner portion 130. In such embodiments, M1macrophages phagocytize the antigen on or in bone matrix 112 and accessinner portion 130 through holes and/or porosity in outer layer 120and/or inner layer 122, thus leaving behind the porous bone matrix 112(e.g., inert beta-tricalcium phosphate, CaCO3, silicon, PLGA,hydroxyapatite, allograft bone, autograft bone, xenograft bone, atitanium implant, a PEEK implant, and/or synthetic bone void filler). Insome embodiments, inner layer 122 consists of a mixture of LPS and LTA.In some embodiments, inner layer 122 consists of a mixture of 50% byweight LPS and 50% by weight LTA. In some embodiments, inner layer 122consists of a mixture of 50% by weight LPS and 50% by weight LTA, mixedtogether at a concentration of 5 micrograms per liter each. In someembodiments, inner layer 122 consists of a mixture of 50% by weight LPSand 50% by weight LTA, mixed together at a concentration of 5 microgramsper liter each, wherein the LPS is derived from E. coli (e.g., E. coliStrain O55:B5) and the LTA is derived from S. aureus. It is recognizedthat 100% LPS or 100% LTA or 100% of any antigen or any combinationthereof can be chosen to optimally activate the innate immune system.

Inner layer 122 may comprise an antigen or antigen mixture (LPS, LTA,and/or other suitable antigen) completely, or inert beta-tricalciumphosphate, CaCO3, silicon, PLGA, and/or hydroxyapatite with the antigenor antigen mixture applied thereon or embedded or injected therein. Insome embodiments, the antigen or antigen mixture (LPS, LTA, and/or othersuitable antigen) is added to implant 110 to create inner layer 122 bydipping implant 110 therein and air drying. In some embodiments, theantigen or antigen mixture (LPS, LTA, and/or other suitable antigen) isadded to implant 110 to create inner layer 122 by placing implant 110(e.g., in the wound or incision) and squirting the antigen or antigenmixture thereon. The antigen or antigen mixture could also be added atany point as part of the 3D printing process in the manufacturing of theimplant.

In some embodiments, inner layer 122 may comprise an infusion ofinterleukins, as discussed above, and is operatively arranged toinitiate the M2 phase of macrophages. In some embodiments, inner layer122 comprises an infusion of capsaicin and/or interleukins. Once the M1macrophages completely and/or partially phagocytose outer layer 120(i.e., all of the LPS, LTA, and/or other suitable antigen is eatenaway), the M1 macrophages encounter inner layer 122, which comprisesinterleukins 4, 10, and/or 13, and are converted to M2 macrophages. TheM2 macrophages begin the reparative process and hence bony repair orfusion. M1 macrophages are converted to M2 macrophages through contactwith inner layer 122 (i.e., the interleukins). Additionally, once the M1macrophages completely and/or partially phagocytize outer layer 120(i.e., the antigen or antigen mixture), interleukins are released by theM1 macrophages to initiate the M2 phase of macrophages. Thus, implant110 can convert M1 macrophages to M2 macrophages in two ways: 1) afterM1 macrophages completely and/or partially phagocytize outer layer 120;and, 2) when M1 macrophages come into contact with inner layer 122. Insome embodiments, inner layer 122 comprises interleukin 4 at 10 ng/ml.

Inner portion 130 may comprise an infusion of interleukins, and asdiscussed above, is operatively arranged to initiate the M2 phase ofmacrophages. In some embodiments, inner portion 130 comprises aninfusion of capsaicin and/or interleukins. Once the M1 macrophagescompletely and/or partially phagocytose outer layer 120 and/or innerlayer 122 (i.e., all of the LPS, LTA, and/or other suitable antigen iseaten away), the M1 macrophages encounter inner portion 130, whichcomprises interleukins 4, 10, and/or 13, and are converted to M2macrophages. The M2 macrophages begin the reparative process and hencebony repair or fusion. M1 macrophages are converted to M2 macrophagesthrough contact with inner portion 130 (i.e., the interleukins).Additionally, once the M1 macrophages completely and/or partiallyphagocytize outer layer 120 and/or inner layer 122 (i.e., the antigen orantigen mixture), interleukins are released by the M1 macrophages toinitiate the M2 phase of macrophages. Thus, implant 110 can convert M1macrophages to M2 macrophages in two ways: 1) after M1 macrophagescompletely and/or partially phagocytize outer layer 120 and/or innerlayer 122; and, 2) when M1 macrophages come into contact with innerportion 130. In some embodiments, inner portion 130 comprisesinterleukin 10 and 13 at 10 ng/ml.

In some embodiments, implant matrix 112 comprises beta-tricalciumphosphate, for example, 3D printed such that inner portion 130 comprisessmall pore sizes and outer layer 120 and inner layer 122 comprise largerpore sizes. The larger pores and reduced density of outer layer 120 andinner layer 122 allows the layers to be infused with the antigen orantigen mixture (LPS, LTA, and/or other suitable antigen) tochemotactically attract M0 and M1 macrophages and monocytes. Theporosity and reduced density of outer layer 120 and inner layer 122 alsospeeds the process of phagocytosis, wherein the M1 macrophagesphagocytose outer layer 120 and/or inner layer 122 until they are goneor partially gone, and only inner portion 130 and/or inner layer 122remain. In some embodiments, and as previously discussed, outer layer120 and/or inner layer 122 are operatively arranged to be partiallyremovable/dissolvable, wherein the M1 macrophages phagocytose theantigen on or in implant matrix 112 and subsequently access innerportion 130 through holes or pores in outer layer 120 and inner layer122. The smaller pores and increased density of inner portion 130, whichcontains the interleukins, slows its dissolution so as to remain untilsuitable bone growth or fusion has occurred. In some embodiments, innerportion(s) 130 comprises a porosity having an average pore size of50-200 microns, outer layers 120 comprises a porosity having an averagepore size of 200-500 microns, and layer 122 comprises a porosity havingan average pore size of 200-500 microns. In some embodiments, the poresof inner portion(s) 130 and outer layers 120 and 122 are interconnectedby channels through implant 110.

Implant 110 generally acts as a dissolving implant or a time releasebone fusion capsule or implant. The M0 macrophages are attracted toimplant 110 and converted to M1 macrophages upon arrival. The M1macrophages “dissolve” or phagocytize outer layer 120 and then innerlayer 122, which comprise the antigen or antigen mixture (LPS, LTA,and/or other suitable antigen) and, once this is done, the M1macrophages encounter the denser inner portion 130 containinginterleukins. Inner portion 130 containing interleukins modulates thetransition of M1 or inflammatory macrophages to M2 or anti-inflammatorymacrophages thereby facilitating the reparative process (i.e., bonegrowth or fusion). Implant 110 is eventually incorporated andtransformed into normal regenerative bone by actively orchestrating thekey cellular actors involved in bone healing.

It will be appreciated that various aspects of the disclosure above andother features and functions, or alternatives thereof, may be desirablycombined into many other different systems or applications. Variouspresently unforeseen or unanticipated alternatives, modifications,variations, or improvements therein may be subsequently made by thoseskilled in the art which are also intended to be encompassed by thefollowing claims.

REFERENCE NUMERALS

-   10 Implant-   12 Implant matrix-   20 Outer portion-   30 Inner portion-   110 Implant-   112 Implant matrix-   120 Layer-   122 Layer-   130 Inner portion

What is claimed is:
 1. An extended release immunomodulatory implantoperatively arranged to facilitate bone morphogenesis, comprising: aninner portion including one or more interleukins; and, an outer portionincluding an antigen operatively arranged to activate an innate immunesystem.
 2. The implant as recited in claim 1, wherein the implantcomprises at least one of inert beta-tricalcium phosphate, calciumcarbonate, silicon, polylactic-co-glycolic acid, and hydroxyapatite. 3.The implant as recited in claim 2, wherein the implant consists of inertbeta-tricalcium phosphate, calcium carbonate, silicon, andpolylactic-co-glycolic acid, wherein the silicon is 1% by weight.
 4. Theimplant as recited in claim 1, wherein the inner portion comprises atleast one of interleukin 4, interleukin 10, and interleukin
 13. 5. Theimplant as recited in claim 1, wherein the antigen comprises at leastone of lipopolysaccharide and lipoteichoic acid.
 6. The implant asrecited in claim 5, wherein the antigen consists of a mixture of: 50% byweight lipopolysaccharide; and, 50% by weight lipoteichoic acid.
 7. Theimplant as recited in claim 5, wherein the antigen consists of 100% byweight lipopolysaccharide.
 8. The implant as recited in claim 5, whereinthe antigen consists of 100% by weight lipoteichoic acid.
 9. The implantas recited in claim 5, wherein the lipopolysaccharide is derived fromEscherichia coli.
 10. The implant as recited in claim 5, wherein thelipoteichoic acid is derived from Staphylococcus aureus.
 11. The implantas recited in claim 1, wherein the inner portion comprises a firstdensity and the outer portion comprises a second density, the firstdensity being greater than the second density.
 12. The implant asrecited in claim 1, wherein the inner portion comprises a first porosityand the outer portion comprises a second porosity, the second porositybeing greater than the first porosity.
 13. The implant as recited inclaim 12, wherein the first porosity and the second porosity areconnected by one or more channels.
 14. The implant as recited in claim1, wherein the inner portion comprises at least one of inertbeta-tricalcium phosphate, calcium carbonate, silicon,polylactic-co-glycolic acid, and hydroxyapatite, impregnated with theone or more interleukins.
 15. The implant as recited in claim 1, whereinthe outer portion comprises at least one of inert beta-tricalciumphosphate, calcium carbonate, silicon, polylactic-co-glycolic acid, andhydroxyapatite, impregnated with the antigen.
 16. The implant as recitedin claim 1, wherein the outer portion consists of the antigen.
 17. Theimplant as recited in claim 1, wherein the inner portion comprises: aninnermost portion including interleukin 10 and interleukin 13; and, aninner layer including interleukin
 4. 18. The implant as recited in claim1, wherein: the inner portion comprises hydroxyapatite; and, the outerportion comprises: an inner layer including beta-tricalcium phosphateincluding one or more interleukins; and, an outer layer including atleast one of lipopolysaccharide, lipoteichoic acid, and interferongamma.
 19. The implant as recited in claim 1, wherein the implantcomprises at least one of allograft bone, autograft bone, xenograftbone, a titanium implant, a polyether ether ketone (PEEK) implant, andsynthetic bone void filler.
 20. An extended release immunomodulatoryimplant operatively arranged to facilitate bone morphogenesis,comprising: an implant matrix including a first material, the implantmatrix including: an inner portion including at least one of interleukin4, interleukin 10, and interleukin 13; and, an outer portion includingat least one of lipopolysaccharide, lipoteichoic acid, and interferongamma.
 21. The implant as recited in claim 20, wherein the firstmaterial comprises at least one of inert beta-tricalcium phosphate,calcium carbonate, silicon, polylactic-co-glycolic acid, andhydroxyapatite.
 22. The implant as recited in claim 21, wherein thefirst material consists of inert beta-tricalcium phosphate, calciumcarbonate, silicon, and polylactic-co-glycolic acid, wherein the siliconis 1% by weight.
 23. The implant as recited in claim 21, wherein theouter portion consists of a mixture of: 50% by weightlipopolysaccharide; and, 50% by weight lipoteichoic acid.
 24. Theimplant as recited in claim 21, wherein the outer portion consists of:100% by weight lipopolysaccharide; or, 100% by weight lipoteichoic acid.25. The implant as recited in claim 21, wherein the inner portion isimpregnated with the at least one of interleukin 4, interleukin 10, andinterleukin
 13. 26. The implant as recited in claim 21, wherein theouter portion is impregnated with the at least one of lipopolysaccharideand lipoteichoic acid.
 27. The implant as recited in claim 20, whereinthe first material comprises at least one of allograft bone, autograftbone, xenograft bone, a titanium implant, a polyether ether ketone(PEEK) implant, and synthetic bone void filler.
 28. A method ofmanufacturing an extended release immunomodulatory implant operativelyarranged to facilitate bone morphogenesis, the method comprising:forming an inner portion of the implant of a first material; applying atleast one of interleukin 4, interleukin 10, and interleukin 13 to theinner portion; forming an outer portion of the implant of a secondmaterial, the outer portion enclosing the inner portion; and, applyingat least one of lipopolysaccharide, lipoteichoic acid, and interferongamma to the outer portion.
 29. The method as recited in claim 28,wherein: the first material comprises at least one of inertbeta-tricalcium phosphate, calcium carbonate, silicon,polylactic-co-glycolic acid, and hydroxyapatite; and, the secondmaterial comprises at least one of inert beta-tricalcium phosphate,calcium carbonate, silicon, polylactic-co-glycolic acid, andhydroxyapatite.
 30. The method as recited in claim 28, wherein the stepof applying the at least one of interleukin 4, interleukin 10, andinterleukin 13 to the inner portion comprises: impregnating the innerportion with the at least one of interleukin 4, interleukin 10, andinterleukin
 13. 31. The method as recited in claim 29, wherein the stepof applying the at least one of interleukin 4, interleukin 10, andinterleukin 13 to the inner portion comprises: creating a substancecomprising the at least one of interleukin 4, interleukin 10, andinterleukin 13; and, applying the substance to the first material. 32.The method as recited in claim 28, wherein the step of applying the atleast one of lipopolysaccharide and lipoteichoic acid to the outerportion to the outer portion comprises: impregnating the outer portionwith the at least one of lipopolysaccharide and lipoteichoic acid. 33.The method as recited in claim 29, wherein the step of applying the atleast one of lipopolysaccharide and lipoteichoic acid to the outerportion to the outer portion comprises: creating a mixture consisting of50% by weight lipopolysaccharide and 50% by weight lipoteichoic acid;and, applying the mixture to the second material.
 34. An extendedrelease immunomodulatory implant operatively arranged to facilitate bonemorphogenesis, comprising: a first component including at least one ofinert beta-tricalcium phosphate, calcium carbonate, silicon,polylactic-co-glycolic acid, hydroxyapatite, allograft bone, autograftbone, titanium, polyether ether ketone; and, a mixture applied to thefirst component, the mixture including at least one oflipopolysaccharide and lipoteichoic acid.
 35. The implant as recited inclaim 34, wherein the first component consists of inert beta-tricalciumphosphate, calcium carbonate, silicon, and polylactic-co-glycolic acid.36. The implant as recited in claim 34, wherein: the mixture is appliedto an outer portion of the first component; and, one or moreinterleukins are applied to an inner portion of the first component. 37.The implant as recited in claim 36, wherein: the mixture consists oflipopolysaccharide and lipoteichoic acid; and, the one or moreinterleukins comprises interleukin 4, interleukin 10, and interleukin13.
 38. The implant as recited in claim 34, wherein: the mixture isapplied to an outer layer of the first component; interleukin 4 isapplied to an inner layer of the first component; and, interleukin 10and 13 is applied to an inner portion of the first component.
 39. Theimplant as recited in claim 34, wherein the first component comprises:an inner portion including hydroxyapatite; an inner layer includingbeta-tricalcium phosphate and one or more interleukins; and, an outerlayer including beta-tricalcium phosphate and the mixture.